Method for detecting a nucleic acid base sequence

ABSTRACT

A method for the detection of diagnostic base sequences in a sample nucleic acid comprises contacting the sample in the presence of appropriate nucleoside triphosphates and an agent for polymerization thereof with a diagnostic primer for the diagnostic base sequence, the primer having a tail sequence comprising a tag region and a detector region such that an extension product of the primer is synthesized when the corresponding base sequence is present in the sample, and any extension product of the diagnostic primer acting as a template for extension of a further primer which hybridizes to a locus at a distance from the diagnostic base sequence. The sample is contacted with a tag primer which selectively hybridizes to the complement of the tag sequence in an extension product of the further primer and is extended, and the presence or absence of the diagnostic base sequence is detected by reference to the detector region in the further primer extension product.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to novel methods for the detection of diagnosticbase sequences in sample nucleic acid. In particular the inventionrelates to the use of tailed primers in such methods.

2. Description of Related Art

The invention is an improvement on currently established procedures forthe detection of nucleic acid sequences. The detection of nucleic acidsequences is a desirable and necessary procedure in the followingexemplary areas; detection and diagnosis of alleles responsible forgenetic diseases in humans and other species; detection and diagnosis ofDNA sequences associated or linked to genes that may or may not beinvolved in disease in humans and other species; detection and diagnosisof neoplasms and the effects of therapy of neoplasms; detection of anddistinction between different pathogens (eg. viruses, bacteria andfungi); determining the purity of animal strains and pedigrees;distinguishing and identifying different humans and animal samples inforensic medicine.

The polymerase chain reaction (PCR) as disclosed for example in U.S.Pat. Nos. 4,683,202 and 4,683,195 has been used to amplify specific DNAsequences. However, PCR does not, by itself, provide a method to detectsingle base mutations. It has been necessary to combine the PCR withother techniques, for example allele specific oligonucleotide probing ofPCR amplification products.

SUMMARY OF THE INVENTION

We have now devised a novel assay system for the detection of diagnosticbase sequences which uses tailed diagnostic primers having a tag regionand a detector region. Under appropriate conditions any diagnosticprimer extension product acts as a template for extension of a furtherprimer. In which case a sequence complementary to the tag region and thedetector region will arise in the further primer extension product. Atag primer is provided which can hybridise to the complement of the tagregion in the further primer extension product and be extended. Adiagnostic base sequence is identified by reference to the sequencecomplementary to the detector region in the tag primer extensionproduct.

Therefore in a first aspect of the present invention we provide a methodfor the detection of a diagnostic base sequence in nucleic acidcomprised in a sample, which method comprises contacting the sampleunder hybridising conditions and in the presence of appropriatenucleoside triphosphates and an agent for polymerisation thereof, with adiagnostic primer for the diagnostic base sequence, the diagnosticprimer having a tail sequence comprising a tag region and a detectorregion, such that an extension product of the diagnostic primer issynthesised when the corresponding diagnostic base sequence is presentin the sample, no extension product being synthesised when thecorresponding diagnostic base sequence is not present in the sample andany extension product of the diagnostic primer acts as template forextension of a further primer which hybridises to a locus at a distancefrom the diagnostic base sequence, and contacting the sample with a tagprimer which selectively hybridises to the complement of the tagsequence in an extension product of the further primer and is extended,and detecting the presence or absence of the diagnostic base sequence byreference to the detector region in the further primer extensionproduct.

The detector region in the further primer extension product may bedetected in a number of ways. For example the sample may be contactedwith detector species capable of emitting a detectable signal uponinteraction with the detector region in the further primer extensionproduct whereby the presence or absence of the diagnostic base sequenceis detected by reference to the detectable signal. It will beappreciated that the detector species cannot become associated with thecorresponding detector region until target dependent hybridisation andfurther primer extension has occurred. This system is well suited forhomogeneous assays and real time or end point analysis. A detectorspecies is any species capable of selective association with thedetector region in a further primer extension product and release of adetectable signal. It will be appreciated that by “selectiveassociation” we mean that the detector species identifies and binds tothe detector region in the further primer extension product to theexclusion of other nucleic acid seqences in the sample. Such detectorspecies may include antibodies and hybridisation probe(s). A particulardetector species is a detector probe such as a labelled hybridisationprobe. Label is conveniently released by the action of for example anexonuclease associated with the polymerase mediated extension of a tagprimer. In the specific description hereinafter we describe a number ofalternative systems. These include detection of the change in shape of aprobe upon hybridisation, the use of two or more probes havinginteractive labels such as for example the use of fluorescence resonanceenergy transfer, the use of scintillation proximity assays (SPA), themeasurement of a change in fluorescence polarisation upon hybridisationof a fluorescently labelled probe. Further systems will be apparent tothe scientist of ordinary skill. These include the use of a solid phasecapture probe for the detector region in the further primer extensionproduct. It will be appreciated that both direct and indirect labellingmethods may be used to detect the immobilised further primer extensionproduct. By way of example a further labelled probe for a region otherthan the detector region may be used. Alternatively, intercalation maybe used to detect the detector region/probe DNA duplex. Also, labelleddNTPs may be incorporated into the further primer extension product.

The sequence of the detector probe need not be the same but isconveniently identical to the sequence of the detector region in thetail. It will be appreciated that minor changes may be made to thesequence of the detector probe without affecting its performance to anysignificant extent.

Alternatively the complement of the detector region is detected byreference to its size contribution to the overall amplification productof the tag and further primers. A convenient size difference may beused, even as little as one base pair difference can be detected on agel. Generally however size differences of at least 5, conveniently atleast 10, at least 15 or at least 20 base pairs are used. This aspect ofthe invention is of particular use where two or more alleles of agenetic locus are to be detected in a single assay mixture.

The tag primer is capable of hybridisation to the complement of the tagsequence in the further primer extension product. It will be understoodthat the diagnostic primer extension product is separated from thefurther primer extension product prior to hybridisation of the tagprimer. The sequence of the tag primer is conveniently identical to thesequence of the tag region in the tail. The tag primer preferablycomprises a sequence capable of hybridisation to all tag sequences. Alltag sequences are preferably identical. Again it will be appreciatedthat minor changes may be made to the sequence of the tag primer withoutaffecting its performance to any significant extent. The use of a commontag primer and common tail sequences has significant cost advantages fora typical assay.

It will be understood that the diagnostic primer tail isnon-complementary to any relevant genomic sequence or adjacent region soas not to compromise the assay.

In known diagnostic PCR procedures mispriming may occur at eachamplification cycle, especially where the primer is used to detect forexample single base mismatches or to detect a particular sequenceagainst a background of related sequences. Such mispriming may onlyoccur as a very low percentage of total priming events per amplificationcycle but will increase significantly as a function of the overallnumber of cycles. The present invention comprises a two stage procedurewherein as a first stage the initial interaction between a diagnosticprimer comprising tag and detector regions and a sample template mayconducted at optimum hybridisation stringency. Any primer extensionproducts are then amplified using a further primer. As a second stagethe above extension products are then amplified using a tag primer andthe further primer. Accordingly, whilst mispriming may still initiallyoccur the overall level may be significantly reduced.

As indicated above the tail sequences may be the same or different butare conveniently identical or substantially similar so that a singletail primer may be used. This facilitates the performance of largemultiplexes without overloading the reaction mix with different primers.We have found that the use of identical tag sequences can beadvantageously used to even out the efficiencies of differentamplification reactions.

We have also found that tailed primers can also be used to prevent theformation of “primer dimers” and other inter-primer artefacts. These area particular problem in homogeneous assays using for exampleintercalating dyes to detect double stranded nucleic acid. They resultin false positive signals. See for example Ishiguro et al, Anal.Biochem., 1995, 229, 207-213, especially pages 211-212. Whilst we do notwish to be limited by theoretical considerations, it is believed thatthe formation of primer dimers is dependent on some degree of homologybetween primers and their use at high concentrations. It may be possibleto reduce the formation of primer dimers by careful primer design.However where many primers are used at high concentrations, for examplein PCR multiplexes, this becomes more difficult. We now disclose the useof diagnostic and further primers which are genome specific at their3′-termini but which carry a detector region and common extensions(tags) at their 5′-termini. These are used in combination with a commontag primer which can prime from the complement of the tag sequence inextension products of further primer(s). Thus whilst primer dimers andother inter-primer artefacts could occur during first phase diagnosticpriming, these cannot be amplified during subsequent rounds of tagspecific priming. It will be appreciated that the diagnostic primers areconveniently used at concentrations which allow satisfactory priming ontheir genomic template(s) but do not allow significant PCRamplification.

The common tag primer is used at higher concentration than the genomespecific primers.

To ensure that primer-dimers and other artefacts are avoided a commontag and common tag primer are preferably used for all primers present ina reaction mix, including control primers.

We have now found that it is advantageous to switch from diagnosticprimer priming to tag primer priming by means of a temperature switch.The primers are selected so that the melting temperature of the tagprimer is higher than the genome complementary region of the diagnosticprimer. An increase in temperature will favour priming, for exampleafter one or conveniently two rounds of diagnostic primer priming, bythe tag primer.

The diagnostic primer may be an allele specific primer. In EP-A-0333465(Baylor College of Medicine) there is described a detection method usingtwo competing primers for the detection of diagnostic base sequenceswhich differ by as little as a single base. This method depends oncareful control of melting temperature (Tm) and is known as competitiveoligonucleotide priming (COP). Competing primers may be used in themethod of this invention, either the primers are differentially labelledor the amplification products are separated according to size, forexample by the use of different size tails on the primers.

Furthermore in our European Patent, Publication No. 0332435, thecontents of which are incorporated herein by reference, we disclose andclaim a method for the selective amplification of template sequenceswhich differ by as little as one base. The above method is now commonlyreferred to as the Amplification Refractory Mutation System (ARMS). Thisis of particular use, for example, where diagnostic base sequencers) areonly present it low concentration in complex nucleic acid mixtures.

Therefore in a preferred aspect of the above detection method a terminalnucleotide of at least one diagnostic primer is either complementary toa suspected variant nucleotide or to the corresponding normalnucleotide, such that an extension product of a diagnostic primer issynthesised when the terminal nucleotide of the diagnostic primer iscomplementary to the corresponding nucleotide in the diagnostic basesequence, no extension product being synthesised when the terminalnucleotide of the diagnostic primer is not complementary to thecorresponding nucleotide in the diagnostic base sequence.

The diagnostic primers for use in the preceding aspect are convenientlydesigned with reference to our above mentioned European Patent,Publication No. 0332435.

By “substantially complementary” we mean that primer sequence need notreflect the exact sequence of the template provided that underhybridising conditions the primers are capable of fulfilling theirstated purpose. This applies equally to diagnostic and tail primers. Ingeneral, mismatched bases are introduced into the primer sequence toprovide altered extension rates with DNA polymerases. Commonly, however,the primers have exact complementarity except in so far asnon-complementary nucleotides may be present at a predetermined primerterminus as hereinbefore described.

In the diagnosis of, for example, cancer the situation may arise wherebyit is desirable to identify a small population of variant cells in abackground of normal cells. The ARMS system is well suited for thispurpose since it discriminates between normal and variant sequences evenwhere the variant sequence comprises a very small fraction of the totalDNA. Whilst we do not wish to be limited by theoretical considerationswe have successfully performed ARMS assays in which the ratio of mutantto normal DNA was 1:100 and we believe that even larger ratios may bereadily used. To optimise the sensitivity of the ARMS reaction it may beperformed in isolation ie. with a single ARMS primer since in duplex ormultiplex reactions there may be competitive interaction between theindividual reactions resulting in a loss of sensitivity. A controlreaction is desirable to ensure that a polymerase chain reaction hastaken place. In a test for an inherited mutation the copy number of themutation and other genomic is typically 1:1 or 1:2, so a genomic controlreaction can be used without compromising sensitivity or creating animbalance in the system. In a cancer test however, the use of a genomiccontrol reaction may swamp the test reaction leading to a loss ofsensitivity. We have now found that ARMS primer(s) comprising tailsequences may advantageously be used in a two stage amplificationprocedure comprising a genomic control reaction. In the first stage ARMSprimer(s) comprising non-complementary tail(s) are used to amplify anyvariant sequence which may be present. In addition to the ARMS reactiona genomic control reaction is performed in the same reaction vesselusing primers at very low concentation. The control reaction primersalso have non-homologous tails which may or may not have the samesequence as the ARMS primer tail(s). In the second stage tail specificprimers are added and the temperature increased to prevent the originalgenomic control primers from functioning. In this second stage anyvariant sequence product is further amplified and the product of thecontrol reaction from the first stage is also amplified to give adetectable product. Thus the ARMS reaction will only take place ifvariant sequence is present in the original sample and the controlreaction will only function if both the first and second stageamplification reactions have worked.

A further and important use of ARMS is for detecting the presence orabsence of more than one suspected variant nucleotide in the samesample. The ability of ARMS to selectively amplify sequences dependingon the predetermined nucleotide sequence of the diagnostic primersenables multiple amplification products to be distinguished simply,accurately and with minimal operator skill thus making it possible toprovide a robust technique for screening a single sample for multiplenucleotide variations. The use of ARMS to detect more than one suspectedvariant nucleotide in the same sample is conveniently referred to asmultiplex ARMS. Multiplex ARMS is thus of particular interest inscreening a single sample of DNA or RNA for a battery of inheritedconditions such as genetic disorders, predispositions and somaticmutations leading to various diseases. Such DNA or RNA may for examplebe extracted from blood or tissue material such as chorionic villi oramniotic cells by a variety of techniques such as those described byManiatis et al, Molecular Cloning (1982), 280-281. Morever as themolecular basis for further inherited conditions becomes known thesefurther conditions may simply be included in the screening technique ofthe present invention.

Multiple amplification products may be distinguished by a variety oftechniques. Thus for example probes may be employed for each suspectedamplified product, each probe carrying a different and distinguishablesignal or residue capable of producing a signal.

A much simpler and preferred method of distinguishing between ARMSamplification products comprises selecting the nucleotide sequences ofthe amplification primers such that the length of each amplified productformed during the process of the present invention is different. In thisregard the number of base pairs present in an amplification product isdictated by the distance apart of the diagnostic and amplificationprimers. Thus the amplification primers may be designed such that eachpotential variant nucleotide is associated with a potentialamplification product of different length.

In an ARMS reaction diagnostic for a particular point mutation thesequence of the primers is largely constrained by the sequence of theDNA adjacent the mutation of interest. The 3′ base of the primer usuallymatches the base altered by the mutation and extra destabilisation isintroduced to give the required level of specificity. The term“specificity”refers to the ratio of the yield of product when an ARMSprimer is used to prime its target sequence compared to the yield ofmis-primed product from the non-target sequence.

In a multiplex ARMS reaction it is desirable that the individual ARMSreactions work with similar efficiency to allow the simultaneousdetection of all the reaction products. This may be achieved for exampleby altering the concentration of the primers, alteration of thenumber/composition of reactions, or alteration of the ammount ofadditional destabilisations introduced into the ARMS primers. Whilstthese methods are normally sufficient to obtain a balanced multiplexARMS reaction the use of tail or tag sequences may have advantages incertain situations. In particular these may allow a more specific test.By way of example, where a strong additional mismatch is used to obtainspecificity the yield of corresponding multiplex product may be low.Reducing the additional mis-match strength may not be possible withoutcompromising specificity. A tail sequence which in combination with atail specific primer provides a good substrate for a DNA polymerase maybe used to balance the multiplex reaction. A range of tail/primercombinations of known priming ability may be provided. Thus by way ofexample as a first amplification step the priming/mis-priming ratio isoptimised without regard to product yield. Product yield is thenbalanced in the second amplification step using an appropriate range oftail/primer combinations.

In our UK Patent No. 2252407 (Zeneca) we disclose and claim thatmultiplex ARMS may be successfully performed where diagnostic primerextension products of more than one diagnostic base sequence of anucleic acid sample comprise a complementary overlap. This unexpectedimprovement to multiplex ARMS is referred to hereinafter as overARMS.OverARMS now facilitates the detection and analysis of, for example,inherited or infectious disease where the potential variant nucleotidesare closely spaced.

In an overARMS reaction the size of the reaction products can be used toidentify individual combinations of variant nucleotides. Where theproducts are separated for example on an agarose gel this approach maybe limited by the resolving power of the gel. By way of example in ahigh resolution agarose gel overARMS may presently be used to identifymutations within about 10-15 bases of each other. The size of the outeroverARMS primer was increased to give a larger product and wesurprisingly found that the yield of the smaller overARMS product wassignificantly reduced. Whilst we do not wish to be limited bytheoretical considerations we believe that target masking takes placedue to the increased Tm of the larger overARMS primer which bindspreferentially to the target DNA and prevents the smaller overARMSprimer from hybridising. Use of a tailed outer overARMS primer mayprovide the increased product size necessary for resolution but since itis non-complementary at its 5′ end the Tm will be similar to the smallerprimer.

OverARMS is conveniently used for HLA typing, in the diagnosis ofβ-thalasaemia, sickle cell anaemia, phenylketonuria (PKU), Factor VIIIand IX blood disorders and α-1-antitrypsin deficieny. A particular usefor OverARMS is in the detection and diagnosis of cystic fibrosis.Convenient cystic fibrosis alleles are disclosed in our European PatentApplication No. 90309420.9; by B. Kerem et al, Science, 1989, 245,1073-1080; by J. R. Riordan et al, Science, 1989, 245, 1066-1073; by J.M. Rommens et al, Science, 1989, 245, 1059-1065; by G. R. Cutting et al,Nature, 346, 366-368; by M. Dean et al, Cell, 61, 863-870; by K.Kobayashi et al, Am. J. Hum. Genet., 1990, 47, 611-615; by B. Kerem etal, Proc. Natl. Acad. Sci. USA, 1990, 87, 8447; by M. Vidaud et al,Human Genetics, 1990, 85, (4), 446-449; and by M. B. White et al,Nature, 344, 665-667.

Our two stage amplification process using diagnostic and tag primers incombination with a further common primer is conveniently carried outusing all three primers simultaneously and preferably using a ratio oftail specific and/or further primer(s) to diagnostic primer(s) of atleast 1:1, such as at least 20:1, at least 30:1, and at least 40:1,preferably at least 50:1.

All of the above detection methods involving PCR amplification may beprovided as homogeneous assays.

The nucleic acid in the sample may be nucleic acid derived from forexample viruses, bacteria (genomes and plasmids), bacteriophages,eukaryotic cells (nuclear, plasmid or organelle), humans, animals,plants, latent viruses in human or other cells. The sample isconveniently obtained from an individual using conventional techniques.The nucleic acid may be DNA, RNA or reverse transcribed RNA. It may benative, fragmented, cloned, degraded, extracted from cells or justreleased upon cell death.

An additional benefit of the method of the invention is in single tubegenotyping ARMS assays. At a poylymorphic locus in a diploid organismthere are typically two different alleles (A and B) and hence threepossible genotypes (AA, AB and BB). One way to determine the genotype isto perform two separate ARMS reactions, one specific for allele A andthe other specific for allele B. It is also possible to include both theA- and B-specific ARMS primers in a single reaction and use differentiallabelling or primer length to determine which of the primers haveamplified. In practice there may be problems such as non-specificitysince the ARMS reaction product from one allele-specific primer may actas a target for mispriming for the other primer. However in the methodof the present invention the initial extension reaction is from thetailed ARMS primer but subsequent amplification is via the tag primer.After, for example the second round of PCR the ARMS primer contributesvery little to the amplification process and consequently theprobability of inappropriate priming from non-target reaction productsis greatly reduced. The use of a temperature shift protocol after saytwo rounds of PCR to promote tag priming, will further reduce the chanceof mis-priming. Detection of the products of different diagnosticprimers is by differential product size or differential labelling.

The method of the invention may be used in combination with a number ofknown detection systems. By way of example it may be used to improve thetaqman assay as described for example by Holland et al, Proc.Natl. Acad.Sci.USA, 1991, 88, 7276-7280 and by Gelfand et al in U.S. Pat. No.5,210,015. A further assay is that described by Yamagata et al in EP-A-0639647. A still further assay is the strand displacement assay (SDA),see for example Walker et al, Nucleic Acids Research, 1996, 24(2),348-353, EP-A-0 678 581, EP-A-0 678 582, and EP-A-0 684 315.

The invention will now be further illustrated but not limited byreference to the following detailed description, Example, Table andFigures wherein:

DESCRIPTION OF THE DRAWINGS

Each of FIGS. 1,2,3, and 4 shows the effect of magnesium concentrationon the fluorescence ratio of FAM/TAMRA in the TagMan embodiment of theinvention.

FIG. 1. The difference between Ya and the No DNA samples is strongest at2.4 mM Mg. However, these conditions also favour ARMS mispriming and thedifference between Ya and Yi is smaller; this is an ARMS dependentevent.

FIGS. 2-4. Each of three replicates for Ya and No DNA is presented forthree different Mg concentrations (FIG. 2—1.2 mM Mg, FIG. 3—2.4 mM Mg,FIG. 4—4.4 mM Mg). The reproducibility is good and the ratios betweenpositive and negative samples is impressive.

FIG. 5 shows a comparison of a TagMan assay of the invention with anoptimised TaqMan assay, looking at an Insulin gene polymorphism). TagManperformed well. Indeed, at 2.4 mM Mg, the difference between positiveand negative results was nearly two-fold better than that for theinsulin amplicon. We believe that the TagMan probe anneals far moreavidly than the amplifying TAG primer.

FIGS. 6-8 show comparisons with various controls. In order to be surethat signals obtained were produced as a result of the introducedreporter portion of the original primer, control experiments usingtailed primers missing this portion were carried out (FIG. 6—1.2 mM Mg,FIG. 7—2.4 mM Mg, FIG. 8—2.4 mM Mg). In all cases, the absence of thereporter region lead to fluorescence ratios comparable to the negativecontrols. No inappropriate probe cleavage was taking place under theconditions tested here.

FIG. 9(a) shows genomic priming using the tailed three phase (3*) primerof the invention. The tag region of the tail (abc) and the detectorregion (xyz) are shown as is the tag primer (abc).

FIG. 9(b) shows complementary strand synthesis from the further primer(cba). A copy of the tailed 3* primer has now been made.

FIG. 10 shows the TagMan detection embodiment of the invention.

FIG. 10(a) shows the further primer extension product (cba and arrow andfollowing dotted line). The tag primer (abc) and probe (xyz) withattached fluorophore and quencher are shown and can now anneal to thecopied tail

FIG. 10(b) shows polymerase mediated extension of the taq primer, thisencounters the hybridised probe and efficiently cleaves the probe,releasing the measured fluorophore away from its quencher. This is thesame as conventional TaqMan.

FIG. 10(c) shows continued amplification of the target region driven bythe tag primer (abc) and efficient cleavage of the TaqMan probe (xyz).This allows real time or end point detection of the releasedfluorophore. The tag primer and the TaqMan probe are included in the PCRat high concentrations, while the long tailed primers are included atlow concentrations, to maximise tag driven priming. In order to maximisethe efficiency of the process, the TaqMan probe should anneal morestrongly than the tag primer, otherwise cleavage will be inefficient.This can be achieved by manipulating the melting temperatures of theprimers and their relative concentrations. When using TaqMan for allelediscrimination the ASO element of the approach requires that the probeannealing is borderline to obtain maximum differentiation between thetwo variants. The new system is more easily optimisable because of theprobe and drive elements are user-selected and can be optimised once forall amplicons.

FIG. 11 shows the Molecular Beacons embodiment of the invention.Molecular beacons makes use of a similar quenching effect: at the endsof a probe are 5 bp sequences complementary to each other. At each endof the probe is a member of a pair of fluorophores, one absorbs theexcitation light and emits it a wavelength which is quenched by theother fluorophore. At low temperatures, the complementary regions of theof the probe cause the formation of the stem loop structure, bringingthe two fluorophores close to each other and amplified target, then itshould hybridise, disrupting the hairpin and releasing the firstfluorophore from the quenching effect of the second.

FIG. 11(a) shows the the further primer extension product (cba and arrowand following dotted line). The tag primer (abc) and molecular beaconsprobe (xyz) can now anneal to the copied tail. Continued amplificationof the target region is driven by the tag primer, the middle portion ofthe primer is copied repeatedly.

FIG. 11(b) shows a further primer extension product resulting from thecontinued amplification. The quenched beacons probe (xyz) hybridises tothe copied middle section of the tag primer. It becomes stretched,releasing the emitting fluorophore from the quenching effect of thesecond fluorophore.

The Molecular Beacons method is a good way to detect amplicons but notso well suited for allele discrimination. In our new scheme, allelediscrimination can be obtained via ARMS but more importantly, the proberegion may be designed with the idea of maximising the stretching outeffect caused by the hybridisation. In particular, the last 5 bp on theends of the probe can be made to hybridise to the target rather than“flapping around”; this should allow the use of shorter probes and yieldstronger signals. There is no need to design and produce new andexpensive probes for each new amplicon, a generic probe can be used formany different amplification targets. The system is multiplexible bychanging the probe and introduced target sequences, and using adifferent pair of matched fluorophores.

FIG. 12 shows the FRET detection embodiment of the invention. The basicmethod for this approach is to introduce two probes each carrying amember of a fluorophore pair. When the two probes hybridise to theiramplified targets (which are essentially adjacent to each other), theabsorbed energy from exciting the first fluorophore is transferred tothe second fluorophore which then emits at its characteristicwavelength. This can be greatly shifted from the excitation and emissionwavelengths of the first fluorophore and produces very low backgrounds.In this format, the spacing between the two probes is crucial andrequires case-by-case optimisation. Furthermore, the use of twofluorescent probes for each amplicon is an expensive and cumbersomepath.

FIG. 12(a) shows continued amplification of the target region driven bythe tag primer (abc) in combination with the further primer (cba). Themiddle portion of the primer (detector region) is copied repeatedly.Also shown are two probes (x,y; and z) which each carry half of anenergy transfer pair.

FIG. 12(a) shows how, after amplification, the two probes hybridise tothe copied middle section of the tag primer. This allows energy transferbetween the probes thus generating a detectable signal, with lowbackground.

By allowing the user to define the probe sites, a single probe pair canbe designed and optimised. This is then suitable for use against anytarget. This system can be multiplexed by simply changing the middleportion of the original primer and using a different pair offluorophores on appropriate probes. A refinement of this technique,suitable for real-tine assays, is to use directly abutting probes which,once hybridised to the introduced target can be ligated together, forexample using a thermostable ligase, and thus fixed in their fluorescingconfiguration. The ligated double probe can then be displaced by the(taq) polymerase. The ligated product may be to be modified to prevent(TaqMan) cleavage of the newly adjoined sequences.

FIG. 13 shows the Capture and Detection embodiment of the invention. Theintroduced middle section which becomes copied can be used as thesequence by which specific PCR products are captured. Post capture, adetector molecule can be introduced by way of a second target relatedprobe and detection proceeds as appropriate. In this way, mutant andnormal amplicons can be decoded, simply by having unique capturesequence.

FIG. 13(a) shows continued amplification of the target region driven bythe tag primer (abc) in combination with the further primer (cba). Themiddle section of the further primer extension product is copiedrepeatedly. Each amplicon may have a characteristic capture signature(ie. xyz can differ).

FIG. 13(b) shows how the middle portion (x′y′z′) of the further primerextension product is a target for capture by an immobilised probe (xyz).A further probe (lmn) which carries a label (eg. biotin, horse raddishperoxidase, or alkaline phosphatase) may be hybridised to the ampliconfor subsequent detection. In this way mutant and normal sequences may bedetected in a single vessel, such as a tube, by using different captureregions to discriminate between the two products.

FIG. 14 shows the Lanthanide Enhanced Genetics System (LEGS) embodimentof the invention. LEGS is disclosed in our PCT patent application no.WO-95/08642. In LEGS, a partially caged Europium ion is attached to aprobe. When a PCR product is rendered single stranded and hybridised tosuch a probe, a double-stranded region is produced. Also present in themixture is a synthetic Intercalator/Sensitiser (I/S) molecule whichintercalates in the double-stranded region. The intercalator also has alinker arm which ends with a second caging group. In an appropriateconformation, the partially caged Eu ion can become fully caged thanksto the I/S molecule. This excludes and leads to a strong, time-resolvedfluorescence effect. The I/S molecule is difficult to synthesise and maynot be fully heat stable.

Using the three phase primer of this invention, the middle phase can beconstructed to allow a probe carrying a chelate of Eu to hybridiseadjacent to a second probe bearing another caging group. This generatesa fully caged Eu ion which then fluoresces. A major advantage of thissystem is that no complex organic synthesis is required. In addition,the second chelator can be introduced in a targeted manner. Using knowntechniques, both the Eu chelated probe and the cage probe can be readilysynthesised.

FIG. 14(a) shows continued amplification of the target region driven bythe tag primer (abc) in combination with the further primer (cba). Themiddle section of the further primer extension product is copiedrepeatedly. The mixture also contains two probes (x,y: and z), one ofwhich carries a chelated lanthanide, the other carries a secondchelating group.

FIG. 14(b) shows how, after amplification, the two probes hybridise tothe copied middle section of the further primer extension product. Thiscauses complete caging of the lanthanide ion resulting in highefficiency time resolved fluorescence.

FIG. 15 shows probe cleavage detection methods other than using the 5′exomculease activity of taq polymerase. The probe and the introducedtarget may differ by one or more bases, rendering any duplex formedbetween the two susceptible to cleavage by a number of methods, such aschemical cleavage or “cleavase” enzyme. Other approaches include theintroduction of a restriction site on the middle position of the primer.After PCR, this may be cleaved releasing the detected fluorophore fromits quencher. The newly synthesised DNA may be restriction endonucleaseresistant (e.g. by using methylated or phosphothioate dNTPs). We thenallow the unprotected probes to be nicked when annealed to the target.Using a thermostable endonuclease would render this assay format fullyhomogeneous and suitable for real time detection. Alternatively, someenzymes require methylated double-stranded DNA for cleavage and these incombination with a methylated probe provide a further detection system.

FIG. 15(a) shows shows the further primer extension product (cba andarrow and following dotted line). The tag primer (abc) and probe (xyz)with attached fluorophore and quencher are shown. These may anneal tothe copied tail.

FIG. 15(b) shows the probe (xyz) being cleaved using chemical orenyzmatic methods.

FIG. 16 shows other binding based methods. The introduced and copied,middle segment of the primer may also be designed as the target for anumber of specific binding techniques, which may be suitable for productdetection. For example, a triple helix motif may be introduced allowingspecific detection of completed double-stranded amplicons. Anotherexample is mismatched probes detected by mismatch binding proteins.Other sequence specific protein binding events may be suitable fordetection of the amplified middle segment. Finally, simple FP probes maybe detected and enhanced by having protein binding superimposed upon theprobe/target hybridisation.

FIG. 16(a) shows continued amplification of the target region driven bythe tag primer (abc) in combination with the further primer (cba). Themiddle section of the further primer extension product is copiedrepeatedly. Each amplicon may have a characteristic capture signature(ie. xyz can differ).

FIG. 16(b) shows how the copy of the middle portion (x′y′z′) of thefurther primer extension product is a target for subsequent binding of anumber of detector molecules. A triple helix is formed using probex*y*z*.

FIG. 17 shows single tube genotyping using differentially labelled 3*ARMS primers.

FIG. 17(a) shows allele specific extension of differentially labelledARMS 3* primers (abcxyz and abcrst) on targets sequences (alleles A andB). Also shown is a common tag primer (abc).

FIG. 17(b) shows that subsequent amplification for both allele A and Bspecific ARMS products is from the common tag primer (abc). Productsfrom the A specific ARMS primer now contain the reporter sequence xyz,whereas products from the B specific ARMS primer contain the reportersequence rst. Differential detection is conveniently by size (xyz andrst are of different lengths) or by signal detection (xyz and rstreporter groups produce different signals).

EXAMPLE 1

Materials and Methods Primers and Probes (Middle) (3′) Genome (5′)Amplifier Reporter Priming Code Comment Portion portion portion S7970Amplifies Tagged 5′CGTACCACGT NONE NONE amplicon GTCGACT3′(SEQ ID NO:14)HpH1F insulin gene 5′AGCAGGTCTG NONE Primer/amplifier TTCCAAGG3′(SEQ IDNO:17) sequence HpH1R insulin gene 5′CTTGGGTGTG NONE Primer/amplifierTAGAAGAAGC3′(SEQ ID NO:18) sequence INS- insulin gene- NONE 5′FAM- NONEBF1 probe CCTGCCTGTCT CCCAGATCAC TAMRA3′(SEQ ID NO:19) S7033 ΔF508common 5′CGTACCACGT NONE 5′CACTAATG primer (Amplifier GTCGACT3′(SEQ IDNO:16) AGTGAACAA tailed) AATTCTCACC ATT3′(SEQ ID NO:20) T2120 TagManProbe NONE 5′FAM- NONE CTGGCATCGG TAGGGTAAGG ATCGGTATCGT AMRA3′(SEQ IDNO:21) T0990 Triple-phase 5′CGTACCACGT 5′(GCGTACT)C 5′GCCTGGCA primerfor GTCGACT3′(SEQ ID NO:16) TGGCATCGGT CCATTAAAG ΔF508 AGGGTAAGGAAAAATATCA amplicon TCGGTATCG3′(SEQ ID NO:22) TTGG3′(SEQ ID NO:23)Bracketed portion is an optional “hinge” region P1292 No reporter target5′(GCGTACT)CG NONE 5′GCCTGGCA primer TACCACGTGTC CCATTAAAG GACT3′(SEQ IDNO:24) AAAATATCA Bracketed portion TTGG3′(SEQ ID NO:23) is an optional“hinge” region

PCRs

1. Insulin amplicon: 25 μl reactions containing

3 mM MgCl₂

200 μM dNTPs

10% (v/v) glycerol

3 ng/ml each of primers HpH1F and HpH1R

50 nM INS-B1F probe

5 μl genomic DNA (or water for negative controls)

0.625 units of Taq polymerase

in 1×Amplitaq Buffer

These reactions were performed using a two step cycle:

40 cycles×{94° C. for 1 min; 58° C. for 2 min}

2. Ya reactions (150 μl) contained 1×ARMS buffer with: 100 μM dNTPs.Primers

T0990 and S7033 at 10 nM, S7970 at 500 nM, T2120 at 50 mM, 225 ng ΔF508

homozygote DNA and 6 unites of Taq Polymerase. Some reactions were

supplemented with Mg to 2.4 or 4.4 mM

Yi reactions were identical to Ya, but the target DNA was a normalhomozygote at the

F508 position.

Other controls contained no target DNA

To control for the reporter sequence in the Triple phase primer, P1292(containing no middle portion) was substituted for T0900.

The PCR cycles were: 2×{94° C. for 1 min}, 62° C. for 2 min, 72° C. for1 min), followed by 40×{94° C. for 1 min, 64° C. for 1 min}.

Analysis

After cycling, an aliquot (10 μl) of each reaction was analysed by gelelectrophoresis to establish the efficiency of amplification.

The remainder was analysed in 100 μl euvettes using the Fluoromaxfluorometer. Where necessary (as in the case of the insulinamplifications), replicate samples were pooled. The excitationwavelength was set to 488 nm and the emission was read at 518 nm (forFAM) and 582 nm (for TAMRA). The ratios were calculated in each case andplotted appropriately.

Results

The optimal [Mg] for the insulin gene region had been established as 3mM (John Todd pers. comm.) Indeed, there was little or no change in thefluoroesence ratio at lower concentrations. This was borne out in thedata shown in FIG. 1. The difference between Ya and the No DNA samplesis strongest at 2.4 mM Mg. However, these conditions also favour ARMSmispriming and the difference between Ya and Yi is less impressive: thisis due to ARMS not TagMan.

Each of three replicates for Ya and no DNA is presented FIGS. 2-4) forthree different Mg concentrations. The reproducibility is good and theratios between positive and negative samples in impressive.

When compared to the results obtained in an optimised TaqMan assay (theInsulin gene polymorphism) the method of this invention performed well(FIG. 5). Indeed, at 2.4 mM Mg, the difference between positive andnegative results was nearly two-fold better than that for the insulinamplicon. This probably reflects the fact that we were able to designthe TagMan probe to anneal far more avidly than the amplifying tagprimer.

In order to be sure that signals obtained were produced as a result ofthe introduced reporter portion of the original primer, controlexperiments using tailed primers missing this portion were carried out(FIGS. 6-8). In all cases, the absence of the reporter region lead tofluorescence ratios comparable to the negative controls. Noinappropriate probe cleavage was taking place under the conditionstested here.

To demonstrate the universality of this approach, a number of furtheramplicons have been worked up to 3* format In each case, the separateallele specific reactions have been successfully combined to permitSingle Tube Genotyping (STG). In one case (factor V Leiden) the assayhas been blind tested extensively on clinical samples (which were typedindependently by a clinical laboratory using a different technology).

1. Cystic Fibrosis Delta F508

Primers (See Table 1):

V663 1: Tag 20a

V6632 common primer, tailed with 20a sequence

V6634: 3* primer, mutant sequence, T2120 reporter

V6634: 3* primer, normal sequence, T4029 reporter

Probes:

T2120: FAMITAMRA labelled,

T4029: TET/TAMRA labelled,

Reaction Mixes:

All reactions were in 1×ARMS buffer with MgCl₂ adjusted to 3.5 mM final,plus ROX internal standard at 60 nM final, and Tag20a at 500 nM.Amplitaq Gold was included at 2 U per 50 μl reaction

Three mixes were typically used

a. Normal only reaction, with primers V6635 and V6632 each at 10 nM,T4029 at 50 nM

b. Mutant only reaction, with primers V6634 and V6632 each at 10 nM,T2120 at 50 nM

c. STG with all three primers included at 10 nM, both probes at 50 nMeach

Cyling Conditions:

20 minutes at 94° C. to activate the Amplitaq Gold

2 cycles of 94° C., 40 s; 62° C., 80 s; 72° C. 40 s (genomic priming)

40 cycles of 94° C, 4 s, 62° C., 80 s (Tag priming)

2. CTLA4A polymorphism

Primers (See Table 1):

V663 1: Tag20a

V356 1: Common primer, tailed with Tag 20a sequence

V6558: 3* primer, mutant specific, carries the TET reporter region(T4029)

V6715: 3* primer, normal specific, carries the FAM reporter region(T2120)

Probes:

T2120: FAM/TAMRA labelled,

T4029: TETITAMRA labelled,

Reaction Conditions:

All reactions were in IXARMS buffer with MgCl, adjusted to 3.5 mM final,plus ROX internal standard at 60 nM final, and Tag20a at 500 nM.Amplitaq Gold was included at 2 U per 50 μl reaction

Three mixes were typically used

a. Normal only reaction, with primers V6715 and V3561 each at 10 nM,T2120 at 50 nM

b. Mutant only reaction, with primers V6558 and V3561 each at 10 nM, T4029 at 50 nM

c. STG with all three primers included at 10 nM, both probes at 50 nMeach

Cycling Conditions:

20 minutes at 94° C. to activate the Amplitaq Gold

3 cycles of 94° C., 40 s; 64° C., 80 s; 72° C., 40 s (genomic priming)

40 cycles of 94° C., 4 s, 64° C., 80 s (Tag priming)

3. BRCA2exon10 polymorphism

Primers (See Table 1):

V6631: Tag 20a

R432-96: common primer, tailed with Tag 20 sequence

V9596: 3* primer, A-variant specific, carries T4029 (TET) reportersequence

W1940: 3* primer, C-variant specific, carries T2120 (FAM) reportersequence

Probes

T2120: FAM/TAMRA labelled,

T4029: TET/TAMRA labelled,

Reaction Conditions:

All reactions were in 1×ARMS buffer with MgCl₂ adjusted to 3.5 mM final,plus ROX internal standard at 60 nM final, and Tag20a at 500 nM.Amplitaq Gold was included at 2 U per 50 μl reaction

Three mixes were typically used

a. “A” only reaction, with primers R432-96 and V9596 each at 25 nM,T4029 at 50 nM

b. “G” only reaction, with primers R432-96 and W1940 each at 25 nM,T2120 at 50nM

c. STG with all three primers included at 25 nM, both probes at 50 nM

Cycling Conditions:

20 minutes at 94° C. to activate the Amplitaq Gold

4 cycles of 94° C., 40 s; 60° C., 80 s; 72° C., 40 s (genomic priming)

45 cycles of 94° C., 40 s, 64° C., 80 s (Tag priming)

4. Factor V Leiden Mutation

Primers (See Table 1):

V0651: 3* primer (wild type sequence) with reporter region correspondingto T2120

V0652: 3* primer (mutant sequence) with reporter region corresponding toT4029

W4085: Tailed common primer

Tag 20a: Driver primer found in the common and the specific primers

Probes:

T2120: FAMITAMRA labelled,

T4029: TET/TAMRA labelled,

Reaction mixes:

All reactions were in 1×ARMS buffer with MgCl₂ adjusted to 3.5 mM final,plus ROX internal standard at 60 nM final, and Tag20a at 500 nM.Amplitaq Gold was included at 2 U per 50 μl reaction

Three mixes were typically used

a. Normal only reaction, with primers W4085 and V0651 each at 25 nM,T2120 at 50 nM

b. Mutant only reaction, with primers W4085 and V0652 each at 25 nM,T4029 at 50 nM

c. STG with all three primers included at 25 nM, both probes at 50 nM

Cycling Conditions:

20 minutes at 94° C. to activate the Amplitaq Gold

3 cycles of 94° C., 41 s; 60° C., 80 s; 72° C., 51 s (genomic priming)

45 cycles of 94° C., 41 s, 66° C., 80 s (Tag priming)

Validation

More than 200 clinical samples have been tested blind with the STG mix.In every case, the results obtained were concordant with those obtainedby clinical collaborators who used PCR and restriction digestion to typethe same samples.

TABLE 1 CODE SEQUENCE V6631 GCGTACTAGCGTACCACGTG (SEQ ID NO:1) T4029CGGTGGACGTGACGGTACGACGAGGCGACG (SEQ ID NO:2) T2120CTGGCATCGGTAGGGTAAGGATCGGTATCG (SEQ ID NO:3) V6632GCGTACTAGCGTACCACGTGCACTAATGAGTGAACAAAATTCTCA (SEQ ID NO:4) CCATT V6635GCGTACTAGCGTACCACGTGTCGACTCGGTGGACGTGACGGTACG (SEQ ID NO:5)ACGAGGCGACGGCCTGGCACCATTAAAGAAAATATCATCTT V6634GCGTACTAGCGTACCACGTGTCGACTCTGGCATCGGTAGGGTAAG (SEQ ID NO:6)GATCGGTATCGGCCTGGCACCATTAAAGAAAATATCATTGG V3561GCGTACTAGCGTACCACGTGATCCTGAAACCCAGCTCAAAT (SEQ ID NO:7) V6558GCGTACTAGCGTACCACGTGTCGACTCGGTGGACGTGACGGTACG (SEQ ID NO:8)ACGAGGCGACGGCGGCACAAATAAAAACTGAACCTGGCTG V6715GCGTACTAGCGTACCACGTGTCGACTCTGGCATCGGTAGGGTAAG (SEQ ID NO:9)GATCGGTATCGGCGGCACAAATAAAAACTGAACCTGGCTA V0652GCGTACTAGCGTACCACGTGTCGACTCGGTGGACGTGACGGTACG (SEQ ID NO:10)ACGAGGCGACGTACTTCAAGGACAAAATACCTGTATTCCAT V0651GCGTACTAGCGTACCACGTGTCGACTCTGGCATCGGTAGGGTAAG (SEQ ID NO:11)GATCGGTATCGTACTTCAAGGACAAAATACCTGTATTCCGC W4085GCGTACTAGCGTACCACGTGCAGGGGAAACCTATACTTATAAGTG (SEQ ID NO:12) GAACATCR432-96 GCGTACTAGCGTACCACGTGAGAAGTTCCAGATATTGCCTGCTT (SEQ ID NO:13)V9596 GCGTACTAGCGTACCACGTGTCGACTCGGTGGACGTGACGGTACG (SEQ ID NO:14)ACGAGGCGACGACTGATCCATTAGATTCAAATGTAGGAA W1940GCGTACTAGCGTACCACGTGTCGACTCTGGCATCGGTAGGGTAAG (SEQ ID NO:15)GATCGGTATCGACTGATCCATTAGATTCAAATGTAGAAC

24 1 20 DNA Homo sapiens 1 gcgtactagc gtaccacgtg 20 2 30 DNA Homosapiens 2 cggtggacgt gacggtacga cgaggcgacg 30 3 30 DNA Homo sapiens 3ctggcatcgg tagggtaagg atcggtatcg 30 4 50 DNA Homo sapiens 4 gcgtactagcgtaccacgtg cactaatgag tgaacaaaat tctcaccatt 50 5 86 DNA Homo sapiens 5gcgtactagc gtaccacgtg tcgactcggt ggacgtgacg gtacgacgag gcgacggcct 60ggcaccatta aagaaaatat catctt 86 6 86 DNA Homo sapiens 6 gcgtactagcgtaccacgtg tcgactctgg catcggtagg gtaaggatcg gtatcggcct 60 ggcaccattaaagaaaatat cattgg 86 7 41 DNA Homo sapiens 7 gcgtactagc gtaccacgtgatcctgaaac ccagctcaaa t 41 8 85 DNA Homo sapiens 8 gcgtactagc gtaccacgtgtcgactcggt ggacgtgacg gtacgacgag gcgacggcgg 60 cacaaataaa aactgaacctggctg 85 9 85 DNA Homo sapiens 9 gcgtactagc gtaccacgtg tcgactctggcatcggtagg gtaaggatcg gtatcggcgg 60 cacaaataaa aactgaacct ggcta 85 10 86DNA Homo sapiens 10 gcgtactagc gtaccacgtg tcgactcggt ggacgtgacggtacgacgag gcgacgtact 60 tcaaggacaa aatacctgta ttccat 86 11 86 DNA Homosapiens 11 gcgtactagc gtaccacgtg tcgactctgg catcggtagg gtaaggatcggtatcgtact 60 tcaaggacaa aatacctgta ttccgc 86 12 52 DNA Homo sapiens 12gcgtactagc gtaccacgtg caggggaaac ctatacttat aagtggaaca tc 52 13 44 DNAHomo sapiens 13 gcgtactagc gtaccacgtg agaagttcca gatattgcct gctt 44 1484 DNA Homo sapiens 14 gcgtactagc gtaccacgtg tcgactcggt ggacgtgacggtacgacgag gcgacgactg 60 atccattaga ttcaaatgta ggaa 84 15 84 DNA Homosapiens 15 gcgtactagc gtaccacgtg tcgactctgg catcggtagg gtaaggatcggtatcgactg 60 atccattaga ttcaaatgta gaac 84 16 17 DNA Homo sapiens 16cgtaccacgt gtcgact 17 17 18 DNA Homo sapiens 17 agcaggtctg ttccaagg 1818 20 DNA Homo sapiens 18 cttgggtgtg tagaagaagc 20 19 26 DNA Homosapiens 19 cctgcctgtc tcccagatca ctamra 26 20 30 DNA Homo sapiens 20cactaatgag tgaacaaaat tctcaccatt 30 21 35 DNA Homo sapiens 21 ctggcatcggtagggtaagg atcggtatcg tamra 35 22 37 DNA Homo sapiens 22 gcgtactctggcatcggtag ggtaaggatc ggtatcg 37 23 30 DNA Homo sapiens 23 gcctggcaccattaaagaaa atatcattgg 30 24 24 DNA Homo sapiens 24 gcgtactcgt accacgtgtcgact 24

What is claimed is:
 1. A method for the detection of a diagnostic basesequence in nucleic acid in a sample, which method comprises contactingthe sample under hybridizing conditions and in the presence ofappropriate nucleoside triphosphates and an agent for polymerizationthereof, with a diagnostic primer for the diagnostic base sequence, thediagnostic primer having a non-complementary tail sequence comprising atag region and a detector region, such that an extension product of thediagnostic primer is synthesised when the corresponding diagnostic basesequence is present in the sample, no extension product beingsynthesised when the corresponding diagnostic base sequence is notpresent in the sample and any extension product of the diagnostic primeracts as a template for extension of a further primer which hybridizes toa locus at a distance from the diagnostic base sequence, and contactingthe sample with a tag primer which selectively hybridizes to thecomplement of the tag region in an extension product of the furtherprimer and is extended, and detecting the presence or absence of thediagnostic base sequence by reference to the detector region in thefurther primer extension product.
 2. A method as claimed in claim 1wherein a detector species is used which selectively associates with thedetector region in the further primer extension product.
 3. A method asclaimed in claim 2 wherein the detector species emits a detectablesignal when cleaved during polymerase mediated extension of the tagprimer.
 4. A method as claimed in claim 2 wherein the detector speciesemits a detectable signal upon selective association with the detectorregion.
 5. A method as claimed in claim 4 wherein the detector speciesis a fluorescently labelled species and the detectable signal arisesfrom a change in fluorescence polarisation upon selective associationwith the detector region.
 6. A method as claimed in claim 2 wherein thedetector species comprises two species each having an interactive label,which labels interact upon selective association with the detectorregion and release a detectable signal.
 7. A method as claimed in claim6 wherein one of the interactive labels is a chelated lanthanide and theother is a chelating group.
 8. A method as claimed in claim 1 whereinthe further primer extension product is captured on a solid phase usinga species which selectively associates with the complement of thedetector region in the further primer extension product.
 9. A method asclaimed claim 1 wherein the detector species comprises a nucleotidesequence identical to the sequence of the detector region in the tail ofthe diagnostic primer.
 10. A method as claimed claim 1 wherein the tagprimer comprises a nucleotide sequence identical to the sequence of thetag region in the tail of the diagnostic primer.
 11. A method as claimedin claim 1 wherein the detector region in the further primer extensionproduct is identified by reference to its size contribution to thefurther primer/tag primer amplification product.
 12. A method as claimedclaim 1 and wherein the further primer has a non-complementary tailsequence comprising a tag region.
 13. A method as claimed claim 1 andwherein the further primer is a diagnostic primer.
 14. A method asclaimed claim 1 wherein the melting temperature of the tag primer ishigher than that of the diagnostic primer so that an increase intemperature provides a switch from diagnostic primer priming to tagprimer priming.
 15. A method as claimed claim 1 wherein more than onediagnostic base sequence is detected in the sample using more than onediagnostic primer, appropriate further primer(s) and tag regions.
 16. Amethod as claimed in claim 15 wherein an identical tag sequence is usedin the tail of all diagnostic primers and/or all further primers.
 17. Amethod as claimed in claim 15 wherein an identical detector region isused in the tail of all diagnostic primers.
 18. A method as claimed inclaim 15 wherein one further primer is used with more than onediagnostic primer.
 19. A method as claimed claim 1 wherein a terminalnucleotide of at least one diagnostic primer is complementary either toa suspected variant nucleotide or to the corresponding normalnucleotide.
 20. A method for the identification of one or more variantdiagnostic base sequences against a background of normal diagnostic basesequences which comprised the use of a method as claimed in claim 1.